The human genome project has succeeded in sequencing most regions of human DNA. Work to identify the genes and sequence alterations associated with disease continues at a rapid pace. Linkage studies are used to associate phenotype with genetic markers such as simple sequence repeats or single nucleotide polymorphisms (SNPs) to identify candidate genes. Sequence alterations including SNPs, insertions, and deletions that cause missense, frameshift, or splicing mutations then may be used to pinpoint the gene and the spectrum of responsible mutations.
However, even when the genetic details become known, it is difficult to use this knowledge in routine medical practice, in large part because the methods to analyze DNA are expensive and complex. When costs are significantly lowered and the methods dramatically simplified, it is expected that DNA analysis will become accessible for use in everyday clinical practice for effective disease detection and better treatment. Ideal DNA analysis is rapid, simple, and inexpensive.
When a disease is caused by a limited number of mutations, or when a few sequence alterations constitute a large proportion of the disease cases, direct genotyping is feasible. Traditional methods range from classical restriction digestion of PCR products to closed-tube fluorescent methods. Closed-tube methods of DNA analysis can be simple to perform. Once PCR is initiated, no further reagent additions or separations are necessary. However, closed-tube methods are traditionally expensive, due in large part to the cost of the fluorescent probes used. Although there are many elegant designs, the probes are often complex with multiple fluorescent dyes and/or functional groups. For example, one popular approach uses a fluorescent dye and a quencher, each covalently attached to an allele-specific probe (1). Two of these “TaqMan®” probes are required to genotype one SNP. Not only are the probes costly, but the time required for hybridization and exonuclease cleavage also limits the speed at which PCR can be performed.
Another example of closed-tube genotyping uses Scorpion® primers, available from DxS Ltd. Originally described in 1999, Scorpion® primers, or “self-probing amplicons,” are formed during PCR from a primer that includes a 5′-extension comprising a probe element, a pair of self complementary stem sequences, a fluorophore/quencher pair, and a blocking monomer to prevent copying the 5′-extension (2). As illustrated in FIG. 1, in the original stem-loop format, the probe element forms the loop, and the stem brings the fluorophore and quencher into close proximity. After PCR, the probe element hybridizes to a portion of the extension product, opening up the stem and separating the fluorophore from the quencher. An additional duplex format, also illustrated in FIG. 1, was later developed in which the fluorophore on the Scorpion® primer is quenched by a quencher on a separate complementary probe that forms a duplex before PCR (3). After PCR, the probe element, which is now part of the amplicon, separates from the quenching probe and hybridizes to the amplicon. In both cases, probing is an intramolecular reaction.
There are several advantages of intramolecular reactions over intermolecular probes. First, intramolecular hybridization is fast and is not a limiting step, even with the current fastest PCR protocols (4). The probe element is stabilized by the intramolecular reaction, increasing probe melting temperatures by about 5-15° C., so that shorter probes can be used, illustratively in areas of high sequence variation. In the stem-loop format, a single oligonucleotide serves both as one of the primers and as a probe. However, such probes can be complex and expensive. The high cost is driven by the high complexity to produce certain probes. For example, each Scorpion® primer requires three modifications to the oligonucleotide primer (a fluorophore, a quencher, and a blocker). A closed-tube genotyping system that retains the advantages of Scorpion® primers, but eliminates their complexity and cost, would be desirable.
Yet another method for genotyping, “Snapback single strand conformation polymorphism, or SSCP”, has been used. SSCP uses a primer of a specific sequence to introduce secondary structure into PCR products that are later separated by electrophoresis to reveal single strand conformation polymorphisms (“SSCP”) (5). In Snapback SSCP, a complementary 8-11 bp primer tail loops back on its complementary sequence in the extension product, creating a hairpin in the single stranded amplicon, which is later detected by gel separation.
As discussed above, Snapback primers may be used to introduce a secondary loop structure into an extension product. However, Snapback primers and other prior art methods discussed herein rely on post-amplification gel separation, or use expensive fluorescently labeled primers. In comparison, the methods of the present invention use a dsDNA dye and melting analysis to monitor hybridization of the hairpin. According to one aspect of the present application, after PCR, illustratively but not limited to asymmetric PCR, intramolecular melting of the hairpin allows genotyping. The intramolecular hybridization is illustrated in FIG. 2. The method is simple because only two PCR primers are required, the only addition being a 5′-tail of nucleotides on at least one primer. No covalent fluorophores, quenchers or blockers are required, greatly reducing the cost of synthesis and assay development. Thus, in one illustrative embodiment, the dsDNA dye is untethered and is free to bind and be released from the nucleic acid solely based on melting.
One issue that has prevented a better method of genotyping revolves around the fact that most genetic diseases are complex. Many different sequence alterations in the same or different genes may contribute to a disease phenotype. The initial hope that most human diseases are caused by a handful of sequence variants has proven not to be true. Many genes can contribute to a particular phenotype, and many different mutations within a gene may cause the same or similar disease patterns. Therefore, to determine the link between a genotype and its resultant phenotype, genetic testing often requires parallel analysis of many coding and regulatory regions. Several methods of screening DNA for abnormalities are available and are known as “scanning” methods. While “genotyping” focuses on detecting specific sequence alterations, mutation scanning can flag the presence of an abnormality, which can then be identified through methods such as genotyping or sequencing.
Sequencing is currently the gold standard for identifying sequence variation. Even though costs are decreasing, sequencing is still a complex process that is not rapid, simple, or inexpensive when applied to specific genetic diagnosis or pharmacogenetics. This remains true for methods that use polonies (6) or emulsion PCR (7). Standard sequencing requires seven steps: 1) amplification by PCR, 2) clean up of the PCR product, 3) addition of cycle sequencing reagents, 4) cycle sequencing for dideoxy termination, 5) clean up of the termination products, 6) separation by capillary electrophoresis, and 7) data analysis. This complexity can be automated and has been in some sequencing centers, but sequencing still remains much more complex than the methods of the present invention. Further, when large or multiple genes are analyzed, over 90% of the sequenced products come back normal. A simple method that could identify normal sequences and common variants would eliminate most of the time, cost, and effort of sequencing.
Snapback primers of the present invention may be used to integrate mutation scanning and genotyping in the same reaction. Scanning may be performed by high-resolution amplicon melting (8) in the same reaction and using the same melting curve as Snapback genotyping. Asymmetric PCR for Snapback genotyping results in two species with different melting transitions, an excess single strand in a hairpin conformation and a double stranded PCR product, preferably with each species melting at a different temperature. Illustratively, the Snapback hairpin will melt at low temperature, and the full-length amplicon will melt at high temperature. The hairpin provides targeted genotyping for common variants, while the full-length amplicon allows scanning for any sequence variant within the PCR product. Similarly, symmetric PCR using two Snapback primers may be used to scan and to genotype two known polymorphisms in one reaction. In a well-characterized gene with precise amplicon melting, it is believed that Snapback genotyping typically can eliminate at least 90% and perhaps as much as 99% of the need for sequencing in the analysis of complex genetic disease.
Combined scanning and genotyping with Snapback primers is attractive because only PCR reagents and a dsDNA dye are needed. No expensive modified oligonucleotides, separations, purifications or reagent addition steps are necessary. Closed-tube analysis eliminates the risk of PCR contamination. Furthermore, Snapback primer annealing is rapid and compatible with the fastest PCR protocols.